The invention claimed herein was made by or on behalf of Universite de Strasbourg, Le Center National De La Recherche Scientifique, AND NexGenix Pharmaceuticals, LLC, who are parties to a joint research agreement signed on Jul. 1, 2007 and related to macrocyclic compounds, such as radicicol and its derivatives, which are useful as kinase and HSP90 inhibitors.
Neurofibromatosis includes two diseases, neurofibromatosis type 1 (NF1) and neurofibromatosis type 2 (NF2). Both NF1 and NF2 are inherited disorders and both encompass mutations which predispose individuals to multiple tumors of the central or peripheral nervous system, and occasionally to other malignancies. Major tumor types associated with NF1 and NF2 involve glial cells (e.g. Schwann cells and astrocytes). Although there is the similarity of the involvement of Schwann cells in NF1 and NF2 tumors, NF1 and NF2 have a spectrum of tumors which involve different types of cells. In addition, NF1 and NF2 are caused by different gene mutations.
Neurofibromatosis Type 2
Neurofibromatosis type 2 (NF2) is a rare form of neurofibromatosis, which is a dominantly inherited tumor suppressor disorder, that affects approximately 1 in 25,000 individuals and is characterized by multiple tumors on the cranial and spinal nerves. NF2 is a different disease from NF1, neurofibromatosis type 1. Although both NF1 and NF2 are tumor predisposition syndromes in the nervous system, the tumor suppressor genes are different and signaling pathways are likely to be different.
Individuals with NF2 are at a high risk for developing brain tumors, in particular tumors on both the seventh and eighth cranial nerves. Bilateral vestibular schwannomas, a type of tumor which occurs on these nerves, occurs in about 95% of affected individuals. Consequently, hearing loss, ringing in the ears, and problems with balance are symptoms frequently associated with NF2.
Schwannomas are tumors consisting of nerve sheath cells or Schwann cells (SCs). Schwann cells support and protect nerve cells and provide nerves with the insulation they need to conduct information. Bilateral vestibular schwannomas, also known as acoustic neuromas, as well as spinal schwannomas and schwannomas of the peripheral nerves are common manifestations of NF2. The symptoms of a schwannoma will depend on its location.
In addition to schwannomas, individuals with NF2 may develop other types of tumors emanating from the nerves, meningeal envelopes, brain and spinal cord. The most common tumor of this type is meningioma; other less common tumors include ependymomas and astrocytomas. Moreover, NF2 patients may have an increased risk for developing mesotheliomas.
Molecular Role of Merlin
NF2 is an autosomal dominant genetic trait, meaning it affects both genders equally and each child of an affected parent has a fifty percent chance of inheriting the gene. NF2 results from a mutation or a deletion of the NF2 gene and is transmitted on chromosome 22 (Sainz et al., 1994, Hum. Mol. Genet. 3: 885-891; Ruttledge et al., 1994, Nat. Genet. 6: 180-184; Rubio et al., 1994, Cancer Res. 54: 45-47; Huynh et al., 1997, J. Neuropathol. Exp. Neurol. 56: 382-390).
The NF2 gene is a tumor suppressor gene that encodes a 595-amino acid protein, termed Merlin. Merlin belongs to the ezrin, radixin, and moesin (ERM) family of proteins (Trofatter et al., 1993, Cell. 75: 826).
Over-expression of Merlin can block both cell proliferation and oncogene-induced transformation (Lutchman and Rouleau, 1995, Cancer Res. 55(11): 2270-2274; Tikoo et al., 1994, J. Biol. Chem. 269(38): 23387-23390). Indeed, Merlin can negatively regulate cyclin D1 levels (Xiao et al., 2002, J. Biol. Chem. 277: 883-886) and loss of Merlin results in overexpression of cyclin D1 (Lallemand et al., 2003, Genes Dev. 17: 1090-1100). However, given its predominant localization to the membrane and cytoskeleton interface, Merlin is not likely to directly control the cell cycle machinery.
The mechanism by which loss of NF2 may contribute to tumor development is not clear. Many studies have focused on this issue using both genetic and biochemical approaches. Several lines of evidence suggest that Merlin can regulate receptor tyrosine kinase activity, trafficking, and degradation. Merlin has been shown to interact directly with the focal adhesion component paxillin in a complex that contains integrin-β1 and ErbB2 (Fernandez-Valle et al., 2002, Nat. Genet. 31(4): 354-362), HGF receptor substrate (HRS) (Scoles et al., 2002, Hum. Mol. Genet. 11(25): 3179-3189; Gutmann et al., 2001, Hum. Mol. Genet. 10(8): 825-834; Soles et al., 2000, Hum. Mol. Genet. 9(11): 1567-1574), and platelet derived growth factor receptor (PDGFR) indirectly through interaction with a PDZ-containing adaptor protein EBP50/NHE-RF (Maudsley et al., 2000, Mol. Cell. Biol. 20(22): 8352-8363; Murthy et al., 1998, J. Biol. Chem. 273(3): 1273-1276). Neuregulin growth factors (EGF family of growth factors), VEGF, and HGF are important mitogens for Schwann cells (SCs) (Krasnoselsky et al., 1994, J. Neurosci. 14:7284-7290; DeClue et al., 2000, J. Clin. Invest. 105(9):1233-1241; Caye-Thomasen et al., 2005, Otol. Neurotol. 26(1):98-101). Neuregulin/ErbB pathways are constitutively activated in human NF2 vestibular schwannomas and inhibitors of these pathways (e.g. antibody against neuregulin and Iressa) block proliferation of NF2-deficient schwannoma cells (Stonecypher et al., 2006, J. Neuropathol. Exp. Neurol. 65:162-175; Hansen et al., 2006, Glia 53:593-600). Recent evidence from Drosophila indicates that Merlin can regulate abundance/turnover of many signaling and adhesion receptors such as Notch, the EGF receptor, Patched, Smoothened, E-cadherin, and Fat. Loss of merlin results in accumulation of these cell surface receptors and activation of the associated signaling pathways (e.g. the EGFR pathway and the Wingless pathway) (Maitra et al., 2006, Curr. Biol. 16(7):702-709).
In addition to cell surface receptors, Merlin has been shown to interact with downstream components of various signaling pathways, including Rac-PAK (p21-activated kinase) pathway. Rac is a member of the Rho family of small GTPases, which organize the actin cytoskeleton and control many cellular processes such as cell proliferation, transformation, and cell motility (Etienne-Manneville and Hall, 2002, Nature. 420(6916): 629-635; Sahai and Marshall, 2002, Nat. Rev. Cancer 2(2): 133-142). PAK can phosphorylate 5518 of Merlin (Xiao et al., 2002, J. Biol. Chem. 277: 883-886; Kissel et al., 2002, J. Biol. Chem. 277(12): 10394-10399) which leads to conformational change and loss of growth-suppressing activity (Shaw et al., 1998, J. Biol. Chem. 273(13): 7757-7764; Shaw et al., 2001, Dev. Cell. 1(1): 63-72). Merlin can also act as a negative regulator of Rac-PAK signaling (Shaw et al., 2001, Dev. Cell. 1:63-72; Kissil et al., 2003, Mol. Cell. 12:841-849; Lallemand et al., 2003, Genes Dev. 17: 1090-1100; Hirokawa et al., 2004, Cancer J. 10: 20-26). Loss of Merlin results in the inappropriate phosphorylation and activation of PAK. Over-expression of Merlin inhibits PAK activation and blocks Rac-induced transformation. (Shaw et al., 2001, Dev. Cell. 1(1): 63-72; Kissil et al., 2003, Mol. Cell. 12(4):841-849). Preliminary evidence indicates that loss of Merlin also leads to activation of the Ras/Raf/Mek/Erk pathway and PI3K-Akt pathway (Rangwala et al., 2005, J. Biol. Chem. 280(12):11790-11797; our preliminary data). A recent study from Drosophila has proposed that Merlin and a related protein expanded function upstream of the Hippo signaling pathway to regulate cell proliferation and apoptosis (Hamaratoglu et al., 2006, Nat. cell biol. 8:27-36; Willecke et al., 2006, Curr Biol. 16(21):2090-2100).
The link between Merlin and growth factor receptor signaling indicates that growth factor receptors may play direct roles in NF2-associated tumor formation and progression. However, possible involvement of Merlin with multiple signaling pathways presents a challenge in developing drugs for the treatment of NF2.
Neurofibromatosis Type 1
NF1 is one of the most common single gene disorder to affect the human nervous system, with an incidence of 1 in 3500 individuals (Sorensen S A, Mulvihill J J, Nielsen A. Ann N Y Acad Sci 1986; 486:30-7.). NF1 affects approximately 1.5 million people worldwide and there is no racial, ethnic, or geographic predilection for the disease. NF1 is an autosomal dominantly inherited genetic disorder with frequent germline deletion or loss-of-function mutations of the NF1 gene, and is caused by mutation in the NF1 gene, which encodes Neurofibromin, a tumor suppressor. Neurofibromin shares a region of similarity with the p120RasGAP protein, therefore functioning as a negative regulator of the Ras pathway. A high spontaneous mutation rate (50%) at the NF1 locus and the substantial variability of its expression ensure that the disorder is unlikely to decrease significantly in the population due to genetic screening.
The signs of NF1 include café-au-lait macules, skin freckling, skeletal defects, learning disability, Lisch nodules, dermal and plexiform neurofibromas (most common), benign tumors of the brain or other organs (e.g. optic pathway astrocytomas, optic neuromas, optic gliomas, cerebral astrocytomas, cerebral gliomas, ganglioneuromas, ependymomas, pheochromocytomas and ganglioneuromas), and malignant neoplasms (e.g. rhabdomyosarcomas, neurofibrosarcomas or malignant peripheral nerve sheath tumors (“MPNST”) or malignant schwannomas) (Korf B R. J Child Neurol 2002; 17(8):573-7; discussion 602-4, 46-51.) Children affected by NF1 also have increased risk for developing a rare form of leukemia-juvenile myelomonocytic leukemia (JMML) (Stiller C A, Chessells J M, Fitchett M. Br J Cancer 1994; 70(5):969-72.). Dermal neurofibromas, subdermal neurofibromas, plexiform neurofibromas and MPNSTs are primarily derived from Schwann cells or their progenitors. Optic gliomas and astrocytomas are derived from astrocytes. Pheochromocytomas are derived from neural crest components (as are neurofibromas and MPNSTs).
The typical characteristic of NF1 is the neurofibroma, of which there are clinically and histologically distinct types. Ninety-five % of patients have discrete benign neurofibromas within the dermis which may develop at any time in life, but their numbers are usually small before puberty. The total number of neurofibromas seen in adults varies from just a few to hundreds or even thousands. These tumors may cause disfigurement, chronic pain and pruritus. Certain patients may develop some of the same disfiguring symptoms that are associated with Elephant Man's disease, a separate disorder originally thought to be NF1. Plexiform neurofibromas may be congenital and are present in 30% of patients with NF1. These tumors represent a major cause of morbidity in NF1. They affect long portions of nerves and infiltrate the nerve and surrounding tissue, resulting in disfiguration and neuralgic complications. In about 2-5% of patients, plexiform neurofibromas transform to malignant peripheral nerve-sheath tumors, which have a significant mortality rate. Although NF1 is usually not a lethal disorder, affected individuals often face a lifetime of morbidity and disfigurement.
The Nf1 gene was identified in 1990 (Wallace et al. 1990 Science 249:181-186; Cawthon et al. 1990 Cell 62:193-201) and its gene product, neurofibromin, is a 250 kD protein of 2818 amino acids that has a catalytic domain related to the GTPase-activating protein (GAP) domain of p120RasGAP (Marchuk et al., 1991 Genomics 11:931-940; Gutmann et al., 1991. Proc. Natl. Acad. Sci. U.S.A. 88: 9658-9662; DeClue et al., 1991. Proc. Natl. Acad. Sci. U.S.A. 88:9914-9918; Martin et al., 1990. Cell 63:843-849; Xu et al., 1990. Cell 63: 835-841; Xu et al., 1990. Cell 62: 599-608). Loss of Nf1 in human neurofibromas, MPNSTs, leukemias, and tumor-derived cell lines results in the elevation of Ras-GTP levels and activation of Ras-Raf-Mek-Erk2 and other MAP kinase pathways (Guha et al., 1996 Oncogene 12: 507-513; Bollag et al., 1996. Nat. Genet. 12:144-148; Basu et al. 1992. Nature 356: 713-715; DeClue et al., 1992 Cell 69:265-273). For example, Ras-GTP levels from a few NF1MPNST-derived cell lines ST88-14, 88-3 and 90-8 are much higher compared to other cell lines with normal neurofibromin. These cell lines also have activated downstream MAP kinase pathways. In addition, cell proliferation and soft agar growth of ST88-14 can be inhibited by injection of an antibody against Ras and expression of the GAP domain of neurofibromin, respectively. Therefore, controlling Ras pathway activity in these cells is important in blocking the transformation properties.
HSP90 and HSP90 Inhibitors/Modulators
In the mid-1950's, it was discovered that phosphorylation can reversibly alter the function of enzymes by means of protein kinases which catalyze phosphorylation, or by protein phosphatases which are involved in the dephosphorylation step. These reactions play an essential role in regulating many cellular processes, especially signaling transduction pathways. In the late 1970's, the Rous sarcoma virus (v-Src)'s transforming factor was discovered to be a protein kinase, and also tumor-promoting phorbol esters were found to be potent activators of protein kinase C, revealing the first known connection between disease and abnormal protein phosphorylation. Since then transduction mechanistic defects have been found to cause numerous oncogenic processes and to have a role in diabetes, inflammatory disorders, and cardiovascular diseases. (T. Hunter, Cell, 100:113-127 (2000); P. Cohen, Nat. Rev. Drug Discov., 1:309 (2002)). Thus selective kinase and phosphatase inhibitors have emerged as important drug targets, and inhibition of kinase phosphorylation activity is one of the most promising strategies for chemotherapy.
Macrocyclic resorcylic acid lactones such as radicicol and the related pochonins, are a structurally related group of secondary metabolites isolated from cultures of the clavicipitaceous hyphomycete Pochonia genus, such as Pochonia chlamydosporia var. catenulate strain P0297. See, e.g., V. Hellwig et al., J. Natural Prod., 66(6):829-837 (2003). These compounds and analogs or derivatives of the compounds have been evaluated as kinase inhibitors or inhibitors of HSP90. Halohydrin and oxime derivatives of radicicol were prepared and evaluated for their v-src tyrosine kinase inhibitory, antiproliferative, and antitumor in vitro activity (T. Agatsuma et al., Bioorg. & Med. Chem., 10(11):3445-3454 (2002).
The heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone whose function is to ensure the proper folding and stability of a number of its client proteins such as kinases and transcription factors (Pearl and Prodromou, 2001 Adv. Protein Chem. 59: 157-186). HSP90 belongs to the ATPase superfamily and consists of three protein domains: the N-terminal ATPase domain, a middle domain responsible for client protein binding, and a C-terminal dimerization domain which also contains a weak ATP-binding domain (Pearl and Prodromou, 2001 Adv Protein Chem. 59: 157-186). Four genes of the HSP90 family are found in humans and their gene products have different cellular locations. The two major cytoplasmic isoforms are HSP90 alpha and HSP90 beta (Hickey et al., 1989, Mol Cell Biol. 9: 2615-2626). Other major isoforms are GRP94 in the endoplasmic reticulum (Argon and Simen, 1999, Semin. Cell Dev. Biol. 10: 495-505) and TRAP1/HSP75 in mitochondria (Felts et al., 2000, J. Biol. Chem. 275: 3305-12). HSP 90 is found to be part of a series of dynamic multiprotein complexes made of co-chaperones including HSP70, HSP40, and Hop. Hydrolysis of ATP causes HSP90 to alter its conformation and allows other co-chaperones such as p23, CDC37, or immunophilins to associate with HSP90 to form a mature complex, which catalyzes the folding and maturation of the client proteins (Pearl & Prodromou, 2000, Curr. Opin. Struct. Biol. 10: 46-51.). The adaptor co-chaperone protein CDC37 mediates interactions between HSP90 and kinases (Pearl, 2005, Curr. Opin. Genet. Dev. 15:55-61; Roe et al., 2004, Cell 116: 87-98.).
Like kinases, heat shock proteins (HSPs) interact with ATP and are important targets for controlling disease, however they have a different mechanistic effect. Immediately after exposure to a stress such as heat, hypoxia or acidosis, cells in most tissues rapidly escalate production rate of the HSPs. It is now believed that heat HSPs are molecular chaperones, i.e., they prevent improper associations and assist in the correct folding of other cellular proteins collectively termed clients and substrates. HSP's are also found in association with tumors and other pathophysiological conditions. In fact, chaperone proteins facilitate the survival of tumor cells in stressful environments by facilitating tolerance of alterations inside the cell. HSPs are ubiquitous, highly conserved among the species, and usually classified by molecular weight to the following major families: HSP100, HSP90, HSP70, HSP60 and small HSPs. These families have structural and functional differences, but work cooperatively at different stages of protein folding. HSP90 has attracted particular attention due to its association with many types of signaling molecules such as v-Src and Raf that play a critical role in malignant transformation and metastasis development. Thus, HSP90 inhibitors are desired for designing chemotherapies, and also for elucidating the interplay in complex signaling networks.
Heat Shock Protein 90's (Hsp90s) are ubiquitous chaperone proteins that maintain the proper conformation of many “client” proteins (see Kamal et. al. Trends Mol. Med. 2004, 10, 283-290; Dymock et. al. Expert Opin. Ther. Patents 2004, 14, 837-847; Isaacs et. al. Cancer Cell, 2003, 3, 213; Maloney et. al. Expert Opin. Biol. Ther. 2002, 2, 3-24 and Richter et. al. J. Cell. Physiol. 2001, 188, 281-290), and are involved in folding, activation and assembly of a wide range of proteins, including key proteins involved in signal transduction, cell cycle control and transcriptional regulation. Researchers have reported that HSP90 chaperone proteins are associated with important signaling proteins, such as steroid hormone receptors and protein kinases, including, e.g., Raf-1, EGFR, v-Src family kinases, Cdk4, and ErbB-2 (Buchner, TIBS, 1999, 24, 136-141; Stepanova et. al., Genes Dev. 1996, 10, 1491-502; Dai et. al., J. Biol. Chem. 1996, 271, 22030-4). Studies further indicate that certain co-chaperones, e.g., Hsp70, p60/Hop/Sti1, Hip, Bag1, HSP40/Hdj2/Hsj1, immunophilins, p23, and p50, may assist HSP90 in its function (see for example Caplan, Trends in Cell Biol., 1999, 9, 262-268). Inhibition of Hsp90 causes these client proteins to adopt aberrant conformations, and these abnormally folded proteins are rapidly eliminated by the cell via ubiquitinylation and proteasome degradation. Interestingly, the list of Hsp90 client proteins includes a series of notorious oncogenes. Four of them are clinically validated cancer targets: HER-2/neu (Herceptin® (trastuzumab)), Bcr-Abl (Gleevec® (imatinib mesylate)), the estrogen receptor (tamoxifen), and the androgen receptor (Casodex® (bicalutamide)), while the others play a critical role in the development of cancer. Some of the most sensitive Hsp90 clients are involved in growth signaling (Raf-1, Akt, cdk4, Src, Bcr-Abl, etc). In contrast, few tumor suppressor genes, if any, seem to be clients of Hsp90 (for lists of client proteins see Pratt et. al. Exp. Biol. Med. 2003, 228, 111-133; Workman et. al. Cancer Lett. 2004, 206, 149-157 and Zhang et. al. J. Mol. Med. 2004, 82, 488-499.), and consequently, inhibition of Hsp90 has an overall anti-proliferative effect. In addition, some client proteins are involved in other fundamental processes of tumorigenesis, namely apoptosis evasion (e.g. Apaf-1, RIP, Akt), immortality (e.g. hTert), angiogenesis (e.g. VEGFR, Flt-3, FAK, HIF-1), and metastasis (c-Met).
The numerous client proteins of HSP90 play a crucial role in growth control, cell survival and development processes, and those clients are known to include receptor tyrosine kinases, serine/threonine kinases, steroid hormone receptors, transcription factors and telomerase. In addition to anti-cancer and antitumorgenic activity, HSP90 inhibitors have also been implicated in a wide variety of other utilities, including use as anti-inflammation agents, anti-infectious disease agents, agents for treating autoimmunity, agents for treating ischemia, and agents useful in treating neurodegenerative diseases and in promoting nerve regeneration (see M. Waza et al, Nature Med. 11:1088 (2005); Rosen et al., WO 02/09696; PCT/US01/23640; Degranco et al., WO 99/51223; PCT/US99/07242; Gold, U.S. Pat. No. 6,210,974 B1). There are reports in the literature that fibrogenetic disorders including but not limited to scleroderma, polymyositis, systemic lupus, rheumatoid arthritis, liver cirrhosis, keloid formation, interstitial nephritis, and pulmonary fibrosis may be treatable. (Strehlow, WO 02/02123; PCT/US01/20578).
There are over one hundred HSP90 client proteins reported in the literature (Solit and Rosen, 2006, Curr. Top. Med. Chem. 6:1205-14). Major HSP90 client proteins include steroid hormone receptors such as the androgen, estrogen and glucocorticoid receptors (AR, ER, and GR) (Whitesell and Cook, 1996, Mol. Endocrinol. 10: 705-712; Segnitz and Gehring, 1997, J Biol. Chem. 272(30):18694-701; Czar et al., 1997 Biochemistry. 1997, 36:7776-85), tyrosine and serine/threonine kinases such as HER2 (ErbB2) (Munster et al., 2002, Cancer Res. 62: 3132-3137.), the insulin-like growth factor-1 receptor (IGF-1R) (Sepp-Lorenzino et al., 1995, J. Biol. Chem. 270: 16580-16587.), Met (Webb et al., 2000, Cancer Res. 60: 342-349.), Flt-3 (Yao et al., 2003, Clin. Cancer Res. 9: 4483-4493.), ZAP70 (Castro et al., 2005, Blood 106: 2506-2512.), Src family kinases (Bijlmakers and Marsh, 2000, Mol Biol Cell. 11:1585-1595.) Raf-1 (Schulte et al., 1995, J. Biol. Chem. 270: 24585-24588.), cyclin-dependent kinases 4 and 6 (Cdk4/6) (Stepanova et al., 1996, Genes Dev. 10: 1491-1502.), MLK3 (Zhang et al., 2004, J. Biol. Chem. 279: 19457-19463.), and Akt (Basso et al., 2002, J. Biol. Chem. 277: 39858-39866.), mutant proteins including v-Src (Xu et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7074-7078; Whitesell et al., 1994, Proc. Natl. Acad. Sci. USA 91:8324-8328), mutant EGFR (Shimamura et al., 2005, Cancer Res. 65: 6401-6408.), mutant B-Raf (da Rocha Dias et al., 2005, Cancer Res. 65: 10686-10691; Grbovic et al., 2006, Proc. Natl. Acad. Sci. USA 103: 57-62.), Bcr-Abl (Gone et al., 2002, Blood 100: 3041-3044; Nimmanapalli et al., 2001, Cancer Res. 61: 1799-1804.) and mutant p53 (Whitesell et al., 1998, Mol. Cell. Biol. 18: 1517-1524.), and other proteins such as HIF-1alpha (Isaacs et al., 2002, J. Biol. Chem. 277: 29936-29944; Mabjeesh et al., 2002, Cancer Res. 62: 2478-2482.), Mdm2 (Peng et al., J. Biol. Chem. 276: 40583-40590.), and HSF-1 (Zou et al., 1998, Cell 94: 471-480.). Many of these HSP90 client proteins are important in controlling cell growth and proliferation, differentiation, and cell survival. This is a putative rationale for the use of HSP90 inhibitors in the treatment of cancer. This subject has been extensively reviewed in the recent literature (Chiosis and Neckers, 2006, ACS. Chem. Biol. 1(5):279-284; Janin, 2005, J. Med. Chem. 48(24):7503-7512; Sharp and Workman, 2006, Adv. Cancer Res. 95:323-348; Solit and Rosen, 2006, Curr. Top. Med. Chem. 6(11):1205-1214). The identification of specific HSP90 clients that are important for the growth of specific cancer types is an active area of research.
Two structurally unrelated natural products, geldanamycin and radicicol, were isolated in 1970 from Streptomyces hygrocopicus and in 1953 from the fungus Monosporium bonorden, respectively. Geldanamycin is a benzoquinone-based ansamycin antibiotic, and radicicol is a macrocyclic lactone antibiotic. They were believed to be kinase inhibitors initially and later found to inhibit HSP90 function via the interaction with the N-terminal ATP binding domain of HSP90 (DeBoer et al., 1970, J. Antibiot. 23: 442-447, Roe et al., 1999, J. Med. Chem. 42:260-266; Schulte and Neckers, 1998, Cancer Chemother. Pharmacol. 42:273-279; Prodromou et al., 1997, Cell 90:65-75; Whitesell et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:8324-8328). More compounds have been found to inhibit the function of HSP90 and are generally referred to as HSP90 inhibitors. Most HSP90 inhibitors inhibit the intrinsic ATPase activity by binding to the N-terminal nucleotide binding site of HSP90 and thus block the formation of the mature complex between HSP90, co-chaperones, and the client proteins since the formation of the mature complex is dependent on ATP hydrolysis. The client proteins are then degraded through the ubiquitin-proteasome degradation pathway (Connell et al., 2001, Nat. Cell Biol. 3: 93-96.). The coumarin antibiotic novobiocin binds to the C-terminal ATP-binding site of HSP90 but with a very weak activity to degrade HSP90 client proteins (Marcu et al., 2000, J. Natl. Cancer Inst. 92:242-248; Marcu et al., 2000, J. Biol. Chem. 275:37181-37186).
Both geldanamycin and radicicol demonstrate good cellular potency but are not suitable for clinical development. Geldanamycin has severe hepatotoxicity and radicicol is not stable in serum thereby having no in vivo anti-tumor activity (Agatsuma et al., 2002, Bioorg. Med. Chem. 10:3445-3454; Soga et al., 2003, Curr. Cancer Drug Targets, 3:359-369). Thus, analogs of radicicol, such as oxime derivatives of radicicol, and analogs of geldanamycin, such as 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), the more water soluble analogue 17-demethoxy, 17-(2-dimethylamino) ethylamino geldanamycin (17-DMAG), and hydroquinone analogue of 17-AAG, have been synthesized and tested. These analogs generally exhibit good efficacy at tolerated doses in in vitro studies and in vivo animal xenograft models (Ikuina et al., 2003, J. Med. Chem., 46:2534-2541; Ge et al., 2006, J. Med. Chem. 49:4606-4615; Maroney et al., 2006, Biochemistry, 45:5678-5685; Shiotsu et al., 2000, Blood, 96(6):2284-2291; Sydor et al., 2006, Proc. Natl. Acad. Sci. U.S.A. 103(46):17408-17413). Several geldanamycin analogs have entered clinical trials for the treatment of cancer. These agents have demonstrated promising results in several types of cancer (e.g. breast cancer, leukemia, melanoma, and etc.).
The clinical experience of 17-AAG has stimulated a search for new HSP90 inhibitors with better pharmacological properties and safety profiles. A number of HSP90 inhibitors in various compound classes have been developed as potential agents for cancer treatment. These include purine-based compounds (PCT publications WO/2006/084030; WO/2002/036075; U.S. Pat. No. 7,138,401; US20050049263; Biamonte et al., 2006, J. Med. Chem. 49:817-828; Chiosis, 2006, Curr. Top. Med. Chem. 6:1183-1191; He et al., 2006, J. Med. Chem. 49:381-390), pyrazole-based compounds (Rowlands et al., 2004, Anal. Biochem. 327:176-183; Dymock et al., 2005, J. Med. Chem. 48:4212-4215; PCT publication WO/2007/021966; WO/2006/039977; WO/2004/096212; WO/2004/056782; WO/2004/050087; WO/2003/055860; U.S. Pat. No. 7,148,228), peptidomimetic shepherdin (Plescia et al., 2005, Cancer Cell 7:457-468; US publication 20060035837), and HSP90 inhibitors in other compound classes (PCT publications WO/2006/123165; WO/2006/109085; WO/2005/028434; U.S. Pat. No. 7,160,885; U.S. Pat. No. 7,138,402; U.S. Pat. No. 7,129,244; US20050256183; US20060167070; US20060223797; WO2006091963). Furthermore, small molecules that modulate HSP functions have been reported. For instance, HDAC inhibitors such as Trichostatin A, SAHA, and FK228 are capable of inhibiting deacetylation of HSP90 and thus modulating the function of HSP90 (Kovacs et al., 2005, Molecular Cell, 18: 601-607). Other molecules modulating the level of HSPs, such as HSP70 and HSP27 may also affect the function of the HSP90 complex (Zaarur et al., 2006, Cancer Res. 66(3):1783-1791). None of these HSP90 inhibitors have previously been shown to inhibit the growth of NF1- or NF2-deficient tumor cells.
Some resorcylic acid lactones have been found to inhibit HSP90, thus natural products radicicol and geldanamycin (P. Delmotte and J. Delmotte-Plaquee, Nature (London), 171:344 (1953); and C. DeBoer et al., J Antibiot (Tokyo), 23:442 (1970), respectively) were shown to suppress the transformed phenotype of cell expressing activated Src (H. J. Kwon et al., Cancer Research, 52:6926 (1992); Y. Uehara et al., Virology, 164:294 (1988)). Related compounds such as herbimycin have been reported to have similar effects (S. Omura et al., J Antibiot (Tokyo), 32:255 (1979).

Other resorcylic acid lactones (RALs) studied in this respect include 17-allylamino-17-demethoxygeldanamycin (17AAG) (D. B. Solit et al., Clin. Cancer Res., 8:986 (2002); L. R. Kelland et al., J. Natl. Cancer Inst., 91:1940 (1999)); 17DMAG (J. L. Eiseman et al., Cancer Chemother. Pharmacol., 55:21-32 (2005)); IPI-504 (J. Ge et al., J. Med. Chem., 49:4606 (2006); oxime derivatives such as KF25706 (S. Soga, et al., Cancer Res., 59:2931 (1999)) and KF55823 (S. Soga et al., Cancer Chemotherapy and Pharmacology, 48:435 (2001)); and Danishefsky et al.'s cycloproparadicicol (A. Rivkin et al., Ibid., 44:2838 (2005)). Structurally related variants include chimeric inhibitors having radicicol's carboxyresorcinol and the geldanamycin's benzoquinone (R. C. Clevenger and B. S. Blagg, Org. Lett., 6:4459 (2004); G. Shen and B. S. Blagg, Ibid. 7:2157 (2004); G. Shen et al., J. Org. Chem., 71:7618 (2006)).

Considerable interest in radicicol's medicinal applications has followed the initial findings. (See U.S. Pat. No. 6,946,456; and U.S. Patent Application Publication Nos. 2003-0211469, 2004-0102458, 2005-0074457, 2005-0261263, 2005-0267087, 2006-0073151, 2006-0251574, 2006-0269618, 2007-0004674, and 2007-0010432).

Strikingly, some resorcylic macrolides that are close analogs of radicicol are known to inhibit kinases but not HSP90. Indeed, LL-Z1640-2 was found to be a potent and selective inhibitor of TAK1 kinase for which radicicol and other resorcylides were not active. (J. Ninomiya-Tsuji et al., J. Biol. Chem., 278:18485 (2003); P. Rawlins et al., Int. J. Immunopharma., 21:799 (1999); K. Takehana et al., Biochem. Biophys. Res. Comm., 257:19 (1999); A. Zhao et al., J. Antibiotics, 52:1086 (1999)). Closely related LL-783,227, where one of the olefins has been reduced, is a potent inhibitor of MEK kinase. (A. Zhao et al., J. Antibiotics 52:1086 (1999)). Compound F87-2509.04 was found to induce degradation of mRNA containing AU-rich elements (ARE) (T. Kastelic et al., Cytokine, 8:751 (1996)) and hypothemycin was found to inhibit the Ras-mediated cellular signaling. (H. Tanaka et al., Jap. J. Cancer Res., 90:1139 (1999)). It has been shown that aigialomycin D is a CDK inhibitor. (S. Barluenga et al., Angew. Chem., Int. Ed., 46(24):3951 (2006)).
Other close analogs of radicicol do inhibit HSP90. Pochonin D is a potent inhibitor of HSP90. (E. Moulin et al., J. Am. Chem. Soc., 127(19):6999 (2005)). And pochonin A has been reported to be a 90 nM inhibitor of HSP90. Pochonin C was found to be an inhibitor of herpes' helicase-primase, which is an ATPase rather than a kinase. (V. Hellwig et al., J. Nat. Prod., 66:829 (2003)). Although radicicol and pochonin C are structurally very similar, they have very different conformations in solution, and different biological activities. (S. Barluenga et al., Chem. Eur. J., 11:4935 (2005). Thus it appears the “floppiness” of the macrocyclic may play an essential role in inhibitory differences among resorcylic acid macrolides, and in any case makes those effects difficult to predict by theoretical methods.
Some resorcylic acid macrolides had been known as kinase or phosphatase inhibitors (U.S. Pat. Nos. 5,674,892; 5,728,726; 5,731,343; and 5,795,910), or to inhibit other enzymes (U.S. Pat. No. 5,710,174 inhibiting FXIIIa catalysis of fibrin cross-linking). Resorcylic acid macrolides were also employed for other medical indications (U.S. Pat. Nos. 3,453,367; 3,965,275; 4,035,504; 4,670,249; 4,778,821; 4,902,711; and 6,635,671).
Radicicol and the pochonins are natural products; intermediates for synthesizing some of their analogues of them may be obtained by fermentation, however relying only upon those natural products or their fermentation derivatives severely limits the range of compounds. Thus a number of novel resorcylic acid macrolides have been synthesized. Many of these are zearalane and related compounds in which the macrocyclic ring contains no carbon-carbon double bond other than between carbons of the phenyl ring. (U.S. Pat. Nos. 3,373,038; 3,586,701; 3,621,036; 3,631,179; 3,687,982; 3,704,249; 3,751,431; 3,764,614; 3,810,918; 3,836,544; 3,852,307; 3,860,616; 3,901,921; 3,901,922; 3,903,115; 3,957,825; 4,042,602; 4,751,239; 4,849,447; and 2005-0256183). Syntheses have also been reported for resorcylic acid macrolides characterized by one double bond between ring carbons outside the phenyl ring. (U.S. Pat. Nos. 3,196,019; 3,551,454; 3,758,511; 3,887,583; 3,925,423; 3,954,805; and 4,088,658). Most of those are 14-member macrocycles, but syntheses have also been reported for the 12-member macrocycle analogs. (U.S. Pat. Nos. 5,710,174; 6,617,348; and 2004-0063778. and PCT publication no. WO 02/48135)
Syntheses have also been reported for radicicol-related compounds having two non-aromatic double bonds and either a halide or a 1,2-oxo group (i.e., an epoxide) on the macrocyclic ring. (U.S. Pat. Nos. 4,228,079; 5,597,846; 5,650,430; 5,977,165; 7,115,651; and Japanese patent document nos. JP 6-279279A, JP 6-298764A, JP 9-202781A, JP 10-265381A2; and JP 2000-236984). Syntheses of oximes of radicicol-related compounds are disclosed in U.S. Pat. Nos. 5,977,165; 6,239,168; 6,316,491; 6,635,662; 2001-0027208; 2004-0053990; Japanese patent document no. JP 2003-113183A2; and PCT publication no. WO 99/55689 Synthesis of cyclopropa-analogs of radicicol is disclosed in U.S. Pat. No. 7,115,651 and PCT Publication No. WO 05/061481. Syntheses of some other resorcylic acid macrolide analogs are disclosed in U.S. patent publication no. 2006-0247448 and in PCT publication no. WO 02/48135. Radicicol as well as Pochonins A and C have also been synthesized. (S. Barluenga et al., Angew. Chemie, 43(26):3467-3470 (2004); S. Barluenga et al., Chemistry—A European Journal, 11(17):4935-4952 (Aug. 19, 2005); E. Moulin et al., et al., Organic Letters, 7(25):5637-5639 (Dec. 8, 2005).
U.S. Pat. No. 7,115,651 to Danishefsky et al., which is incorporated by reference herein in its entirety, describes derivatives of radicicol, including cyclopropyl analogs, and the use of these compounds as therapeutic agents.
International Publication No. WO 2008/021213 to Winssinger et al., which is incorporated by reference herein in its entirety, describes certain analogs and derivatives of radicicol and pochonins useful as inhibitors of HSP90, including pharmaceutical compositions comprising the compounds and methods for the treatment of various diseases mediated by HSP90.
International Publication No. WO 2008/150302 to Nexgenix Pharmaceuticals, which is incorporated by reference herein in its entirety, describes uses and methods for the treatment of neurofibromatosis with analogs and derivatives of radicicol and pochonins.
Despite the progress described above, chemical biologists continue to suffer from a limited ability to knock out specific kinase activity in order to deconvolute the role of specific kinases within complex signaling networks. Small molecules that can permeate cells have promise for solving this problem. And it has become increasingly apparent that the biological function of kinases is often regulated by their conformation, which is in turn dictated by their phosphorylation level and by intra- and inter-molecular associations. Small molecule inhibitors also have the potential to discriminate between different conformations of a given kinase, thus small molecules offer a means to dissect the respective functions of those conformation. Unfortunately the portfolio of known kinase inhibitors cannot yet support the full range of work to be done in parsing the roles of the various members of the kinome. This is not a merely academic pursuit, because the rationality of drug design will continue to suffer until kinase mechanisms and their selectivity is understood.
Current treatments for NF2- and NF1-associated tumors consist of surgical removal and focused-beam radiation. Neither treatment is considered optimal. Most patients with NF2 and NF1 require multiple surgical and/or focused beam radiation procedures during their lifetime. Since the tumors of NF2 most frequently lie on nerves near the brain and spinal cord, their surgical removal is not without risk. For instance, surgical removal of vestibular schwannomas typically results in complete hearing loss and frequent facial nerve damage. Focused-beam radiation also has a significant incidence of hearing loss and facial nerve damage. Accordingly, a strong need exists for safer treatment options for NF2 and associated tumors (e.g. schwannomas, meningiomas, and mesotheliomas) and NF1 and associated tumors (e.g. dermal neurofibromas, subdermal neurofibromas, plexiform neurofibromas, MPNSTs, gliomas, astrocytomas, and JMML). The present invention provides a novel method for treating NF2 and related NF2-deficient tumors and NF1 and related NF1-deficient tumors and their associated signs and symptoms by administering radicicol or its derivatives.